Abstract:

An object of the present invention is to provide a method utilizing a
carrier for measuring hyaluronic acid wherein deposition of carrier is
less, a storage stability is good and an accuracy in measurement has same
high degree as that performed by the conventional reagents, and a reagent
kit for the same. Also, the present invention relates to "a method for
measuring hyaluronic acid, comprising forming a hyaluronic acid/HABP
complex by contacting hyaluronic acid in a sample with HABP, reacting
said complex with an anti-HABP antibody-supported carrier, measuring
optical change by agglutination product generated by said reaction and
calculating the quantity of hyaluronic acid from the measured value" and
"a reagent kit for the measurement of hyaluronic acid comprising a
reagent comprising a hyaluronic acid binding protein and a reagent
comprising an anti-hyaluronic acid binding protein antibody-supported
carrier".

Claims:

1-5. (canceled)

6. A reagent kit for the measurement of hyaluronic acid comprising a
reagent comprising a hyaluronic acid binding protein and a reagent
comprising an antibody to hyaluronic acid binding protein supported on a
carrier by physical adsorption.

7. The reagent kit according to claim 1, wherein the carrier is latex
particles.

8. The reagent kit according to claim 2, wherein the average diameter of
the latex particles is 0.05 to 0.3 μm.

9. The reagent kit according to claim 1, wherein the hyaluronic acid
binding protein is selected from proteoglycan, link protein, and
hyaluronectin.

Description:

TECHNICAL FIELD

[0001]The present invention relates to a method for measuring hyaluronic
acid having good storage stability and affording high degree of accuracy
in measurement and a reagent kit for the same.

BACKGROUND ART

[0002]Hyaluronic acid is present mainly in the synovial fluid and ocular
vitreous humor of animal and connective tissue such as the umbilical cord
and the upper dermis of animal. The concentration of hyaluronic acid in
the blood is known to increase in the patients with rheumatoid arthritis,
cancer and liver disease and known as a useful marker for diagnosis of
these diseases, and therefore, various methods for measuring hyaluronic
acid have been developed to date.

[0003]On the other hand, for immunological measurement, methods of
utilizing latex particles have been used widely because of its simple
procedure and applicability to multipurpose measuring devices. The method
employing latex particles as a reagent for measuring hyaluronic acid has
been described, for example, in JP-B-3424504. In the aforementioned
patent publication, a method comprising processes of supporting a
hyaluronic acid binding protein on carrier particles, forming a reaction
complex between the particles and hyaluronic acid in a sample and
determining the hyaluronic acid by detecting the reaction complex has
been described. In Examples of the aforementioned patent publication,
experiments have been carried out using latex particles with average
particle size of 368 nm in diameter. However, when such a particles is
used as a reagent, usually, the latex particles has to be dispersed by
shaking or the like before use, because the latex particles with average
particle size of 300 nm (0.3 μm) or larger in diameter have a tendency
to be easily deposited. Therefore, for the measurement of hyaluronic acid
using a carrier such as latex particles, the development of a reagent,
which is not deposited easily and does not need a cumbersome treatment
such as shaking before use, has been desired.

[0004]Patent Literature 1: JP-B-3424504

DISCLOSURE OF THE INVENTION

Problem to be Solved by the Invention

[0005]On the view of above situation, an object of the present invention
is to provide a method utilizing a carrier for measuring hyaluronic acid
wherein deposition of carrier is less, a storage stability is good and an
accuracy in measurement has same high degree as that performed by the
conventional reagents, and a reagent kit for the same.

Means for Solving the Problem

[0006]To store a reagent containing latex particles for the measurement of
hyaluronic acid in more stable, the present inventors have investigated
to find a method of supporting a hyaluronic acid binding protein
(hereinafter, optionally referred to as HABP) on latex particles with
less deposition (average particle size: 0.3 μm or less in diameter) by
chemical bond or physical adsorption. However, it was difficult to
efficiently support HABP on latex particles, and therefore, measurement
could not be performed with a high degree of accuracy. Then, as the
results of intensive study, the inventors have found that agglutination
reaction of latex particles in accordance with the quantity of hyaluronic
acid can be induced effectively by the reaction of latex particles, which
have preliminarily been sensitized with a monoclonal antibody for HABP,
with a complex formed between HABP and hyaluronic acid. Further, the
present inventors have found that a reagent for the measurement of
hyaluronic acid with good storage stability can be prepared by storing
the latex particles which was preliminarily sensitized with a monoclonal
antibody for HABP and the hyaluronic acid binding protein individually as
separate reagents. Furthermore, they have found that, as described above,
the step can be proceeded by forming a complex preliminarily by reacting
hyaluronic acid with HABP and then by reacting the complex with the latex
particles sensitized with a monoclonal antibody for HABP, and thus
completed the present invention.

[0007]That is, the present invention relates to "a method for measuring
hyaluronic acid comprising forming a hyaluronic acid/HABP complex by
contacting hyaluronic acid in a sample with HABP, reacting said complex
with an anti-HABP antibody supported carrier, measuring optical change of
agglutination product generated by said reaction, and calculating the
quantity of hyaluronic acid from the measured value" and "a reagent kit
for the measurement of hyaluronic acid comprising a reagent comprising
HABP and a reagent comprising an anti-HABP antibody supported carrier".

EFFECT OF THE INVENTION

[0008]According to the measurement method of the present invention, in
comparison with conventional method, the measurement can be performed by
simple procedure such as no need for shaking of the reagent before use,
and also, measurement can be performed with same high degree of accuracy
as that by conventional reagents. In addition, the reagent kit of the
present invention provides less deposition of carrier and good storage
stability; in addition, using said reagent kit, measurement of hyaluronic
acid can be achieved with same degree of accuracy as that by conventional
reagents

BEST MODE FOR CARRYING OUT THE INVENTION

[0009]As the hyaluronic acid binding protein (HABP) in regard to the
present invention, any one containing hyaluronic acid binding region of
proteins which have property to bind hyaluronic acid such as
proteoglycan, link protein, hyaluronectin and the like may be adopted
without specific limitation, and it may include above described protein
itself, a partial protein containing hyaluronic acid binding region of
the above described protein, a substance containing such partial protein
and a recombination protein produced by incorporating a gene fragment
encoding hyaluronic acid binding region of the above described protein
into other protein or the like.

[0010]As an anti-HABP antibody in regard to the present invention, any one
of antibody against HABP whether it is monoclonal or polyclonal may be
adopted. A polyclonal or a monoclonal antibody purified affinity
chromatography with single epi tope is preferable, and a monoclonal
antibody capable of binding efficiently with hyaluronic acid is
particularly preferable. Among these, the use of Fab, Fab', F(ab')2
and the like produced by appropriate digestion of these antibodies using
an enzyme such as pepsin and papain is preferable. When polyclonal
antibody is used as anti-HABP antibody, the antibody can be prepared by a
conventional method of immunization of an animal such as horse, cow,
sheep, rabbit, goat, rat or mouse with hyaluronic acid binding protein
according to the methods described, for example, in "Matsuhashi, T. et
al., Introduction to Experimental Immunology, 2nd ed., 1981, Japan
Scientific Societies Press". When monoclonal antibody is used as
anti-HABP antibody, the antibody can be prepared according to a
conventional method, namely, the cell fusion technology established by
Kohler and Milstein (G. Kohler and C. Milstein: Nature, 256, 495 (1975)),
for example, using a hybridoma cell obtained by fusing a cell line
derived from mouse myeloma with cells from the mouse spleen which is
preliminarily immunized with hyaluronic acid binding protein.

[0011]As a carrier in regard to the present invention, any one of carriers
usually used in the immunological measurement can be adopted,
specifically as preferable ones, carriers prepared from, for example,
natural organic polymer substances such as red blood cell, bacteria, cell
fragment and the like, assembly of molecule such as liposome, polymeric
micelle and the like, synthetic polymer compounds such as polystyrene,
polyacrylic acid, polymethacrylic acid, polyacrylamide,
polyglycidylmethacrylate, polypropylene, polyvinylchloride, polyethylene,
polychlorocarbonate, silicone resin, silicone rubber and the like,
inorganic substances such as porous glass, ground glass, alumina, silica
gel, activated carbon and metal oxide are included. In addition, these
carriers can be used in various forms such as tube, bead, disc type chip,
micro particle or latex particle. Among them, the latex particle is
particularly preferable from the points, for example, that chemical
treatment of the surface of the carrier can be easily carried out as
appropriately for any purposes because the carrier material is artificial
polymer and that nonspecific reaction hardly takes place. As to the
quality of material, it has no specific limitation, but preferably
includes, for example, styrene type latex particle such as polystyrene
latex particle and acrylic acid type latex particle.

[0012]In this connection, among these latex particles, polystyrene latex
particle and the like which are prepared by emulsion polymerization
reaction without use of emulsifying agent are particularly preferable.
Because, as they have a surface with strong hydrophobic nature, proteins
or peptides can be adsorbed smoothly, and also as they have negatively
charged surface and cause mutual repulsion between them, and they can
stably disperse in a solution even in the absence of emulsifying agent.
In addition, various modified latex particle (for example, a carboxylic
acid modified latex particle produced by introducing carboxyl group into
the above described polystyrene), a magnetic latex particle (a magnetic
particle-encapsulated latex particle) and the like can be used as well.

[0013]In addition, as for latex particles to be used in the present
invention, commercially available latex with small average particle
diameter, namely, with large surface area per unit weight, is able to
support antibody efficiently and also provides a good storage stability
(good dispersibility in a solution), and so, it is used preferably. In
more specifically, the average particle diameter is usually 0.05 to 0.3
μm, preferably 0.1 to 0.25 μm. Using such latex particles with
small average diameter, deposition of the particle can be avoided and it
can be achieved to efficiently support anti-HABP antibody on the latex
particles. That is, by the use of such anti-HABP antibody-supported latex
particles, both the increased stability of the measurement reagent and
high accuracy of measurement can be achieved.

[0014]The method of supporting an anti-HABP antibody involved in the
present invention on a carrier involved in the present invention may be
performed without specific limitation by contacting the anti-HABP
antibody with the carrier. All the supporting methods well known per se
usually used in this field can be included, and for example, as an
exemplary method, method of supporting the anti-HABP antibody on the
carrier by physical adsorption, so called physical adsorption method
(refer to, JP-A-1993-41946; SUMILON Technical Report, SUMILON ELISA
series 1 Introduction to ELISA Method, published by Sumitomo Bakelite
Co., Ltd.; SUMILON Technical Report, SUMILON ELISA series 2 Solid Phase
Surface of ELISA Products, published by Sumitomo Bakelite Co., Ltd., and
so on) is included as representative examples. The aforementioned method
is usually used as a preferable method when, for example, synthetic
polymer compounds such as polystyrene, polypropylene, polyvinylchloride,
polyethylene, polychlorocarbonate and the like; activated carbon;
inorganic substances such as porous glass, ground glass, alumina, silica
gel, metal oxide hydroxy apatite and the like are used as a carrier.
Among them, it is particularly preferable when glass, polystyrene,
polyvinylchloride and the like are used in the form of, for example,
tube, bead, disc chip, micro particle or latex particle.

[0015]Taking a case as an example when an anti-HABP antibody involved in
the present invention is supported on the latex particles, the latex
particles are added so as to be concentration of usually 0.1 to 10%
(w/v), preferably 0.2 to 5% (w/v) and suspended in a solvent such as a
buffer solution containing usually 0.05 to 2 mg/ml, preferably 0.1 to 1
mg/ml of an anti-HABP antibody involved in the present invention, and
after reacting usually at 5 to 30° C. and usually for 2 to 3
hours, then, post treatments usually conducted in this field such as, for
example, centrifugation, blocking treatment using a solution containing
an appropriate protein such as bovine serum albumin (BSA) are carried
out, and thus work up the supporting process. In this connection, it can
also be achieved to support the anti-HABP antibody on a carrier by
chemical binding methods usually used in this field.

[0016]The method for measuring hyaluronic acid of the present invention
may be performed by the process wherein a hyaluronic acid/HABP complex is
formed by contacting hyaluronic acid in a sample with HABP, followed by
reacting the said complex with anti-HABP antibody supported carrier,
followed by measuring optical change of agglutination product generated
by the said reaction and followed by calculating the quantity of
hyaluronic acid from the measured value.

[0017]In this connection, the measurement of the optical change described
herein means the measurement of the optical change caused by the
formation of immunoagglutination, and more specifically, in this
category, immunoagglutination methods such as reversed passive
agglutination method, nephelometric immunoassay and turbidimetric
immunoassay are included. These measurement methods may be performed
according to the method well known per se. When the reversed passive
agglutination method is to be employed, the method may be carried out
according to the procedure described, for example, in "Successive Course
on Biochemical Experiment 5: Investigative Approach to
Immunobiochemistry", Tokyo Kagaku Dojin Co., Ltd., pp. 36-37, "A Manual
of Clinical Laboratory Method", 30th ed., Kanehara & Co., Ltd., pp.
844-845, and when nephelometric immunoassay is to be employed, the method
may be carried out according to the procedure described, for example, in
"A Manual of Clinical Laboratory Test", 30th ed., Kanehara & Co.,
Ltd., pp. 851-853, and when turbidimetric immunoassay is to be employed,
the method may be carried out according to the procedure described, for
example, in "A Manual of Clinical Laboratory Method", 30th ed.,
Kanehara & Co., Ltd., pp. 853-854.

[0018]Taking the turbidimetric immunoassay using latex particle carrier as
an example, the measuring method of the present invention will be
described more specifically below. That is, a sample containing
hyaluronic acid (more specifically, for example, body fluid such as
blood, plasma, serum, synovial fluid, pleural fluid, lymph fluid, spinal
fluid and urine) is contacted and mixed with a reagent containing above
described HABP to form hyaluronic acid/HABP complex. Then, for example, a
reagent, wherein above described anti-HABP antibody is supported
(sensitized) on latex particles with average particle diameter of, for
example, 0.05 to 0.3 μm, preferably 0.1 to 0.25 μm, is reacted with
above described complex. The degree of resulting agglutination is
measured, for example, by means of absorbance, and the concentration is
determined from a calibration curve preliminarily prepared using standard
sample, and thus, the quantity of hyaluronic acid in a sample is assayed.
In this connection, absorbance measurement may be carried out usually at
wavelength of 340 to 1000 nm, preferably at 500 to 900 nm. In addition,
determination of the degree of agglutination is not limited to the
measurement of absorbance; the degree may be measured by any one of the
methods well known per se, for example, by nephelometry or by counting
immunoassay. In addition, when the hyaluronic acid/HABP complex is
reacted with a reagent containing an anti-HASP antibody which has been
supported on a carrier such as latex particles (hereinafter, may referred
to as anti-HABP antibody-supported carrier), appropriate agglutination
accelerating agent may be added. In this connection, the specific example
of such agglutination-accelerating agent will be described in the section
of "reagent kit" in the present invention.

[0019]In the measurement method of the present invention, the use
concentration of HABP in HABP reactions is, although it may vary
depending on the detection limit of hyaluronic acid be set out, usually,
equal to or more of the concentration which is capable of binding with
all amount of hyaluronic acid corresponding to the set concentration of
detection limit, preferably 5 times or more, more preferably 10 times or
more of the set concentration of determination limit. In this connection,
the upper limit concentration of hyaluronic acid on this occasion has no
limitation, in consideration of economical amount of the hyaluronic acid,
the concentration is usually 50,000 times or less, preferably 10,000
times or less. Specifically, the concentration is usually from 0.1 to
1000 μg/ml, preferably from 0.5 to 1000 μg/ml, and more preferably
from 0.5 to 100 μg/ml. For example, when hyaluronic acid concentration
in serum is measured, as usual determination limit is from 10 to 1000
ng/ml, the use concentration of HABP in HABP reaction may, therefore, be
set out appropriately within the above-described range based on the
determination limit.

[0020]In addition, as to the pH in the aforementioned reaction, the range
thereof is not specifically limited as long as it does not inhibit
formation of the complex, and is usually 5 to 10, preferably 6 to 8.
Also, as to the temperature in the aforementioned reaction, the range
thereof is not specifically limited as long as it does not inhibit the
formation of the complex, and is usually 5 to 40° C. In addition,
as the reaction time may differ according to the reaction condition such
as HABP used and pH and temperature, the reaction may be conducted for
several seconds to several hours as appropriate according to each
condition.

[0021]In the measurement method of the present invention, the use
concentration of anti-HABP antibody-supported carrier in the reaction
between anti-HABP antibody-supported carrier and hyaluronic acid/HABP
complex is, while it may vary depending on the use concentration of HABP
in the above reaction, usually 0.2 to 25 mg/ml, preferably 0.5 to 12
mg/ml when latex particles having 0.01 to 0.1 mg/mg of supporting amount
of anti-HABP antibody is used, and, if it is within the range of
aforementioned concentration, the hyaluronic acid in a sample can be
measured with a high degree of accuracy. In this connection, the
condition and the time for the reaction of anti-HABP antibody-supported
carrier with hyaluronic acid/HABP complex may be adopted in accordance
with that for the above mentioned HABP reaction.

[0022]As a reagent kit for the measurement of hyaluronic acid of the
present invention, a kit comprising both a reagent comprising HABP and a
reagent comprising anti-HABP antibody-supported carrier may be included.
In this connection, the aforementioned kit may contain a standard
substance usually used in this field such as, for example, potassium
hyaluronate (derived from cockscomb, produced by Wako Pure Chemical
Industries, Ltd.) and sodium hyaluronate (derived from Streptococcus
species, produced by Wako Pure Chemical Industries, Ltd.).

[0023]The reagent comprising HABP in the reagent kit for the measurement
of hyaluronic acid of the present invention may be any one of reagent
comprising HABP as described above, which may be dissolved in an
appropriate buffer solution. As the buffering agents used for this
purpose, any kind of buffering agent usually used in the immunological
measurement, for example, Tris buffering agent, phosphate buffering
agent, veronal buffering agent, boric acid buffering agent and Good
buffering agent can be adopted, and the concentration of such buffering
agent is usually 5 to 300 mM, preferably 10 to 150 mM, and the pH is
usually 5 to 10, preferably 6 to 8, and the concentration and pH are each
selected appropriately from above described corresponding range.

[0024]As to the concentration of HABP in the above reagent comprising
HABP, while it may vary depending on the kind of HABP used, the
concentration in the reaction may be set out to be the same concentration
as described above, and may be selected appropriately so as to be within
the range from 0.1 to 500 μg/ml, preferably 0.5 to 100 μg/ml.

[0025]In a reagent kit for the measurement of hyaluronic acid of the
present invention, a reagent comprising an anti-HABP antibody-supported
carrier may be any one of reagent containing above described anti-HABP
antibody-supported carrier, which may be a suspension of anti-HABP
antibody-supported carrier in an appropriate buffer solution or a
lyophilized product thereof. As the buffering agents used for this
purpose, any kind of buffering agent is used as long as not to inhibit
binding between anti-HABP antibody involved in the present invention and
HABP, and includes the same buffering agents as used for the above
described reagent comprising HABP, and also, the pH and the concentration
may be net out by the same way according to the above described value.

[0026]In addition, the reagent comprising an anti-HABP antibody-supported
carrier is provided in many cases in the form of suspension suspended in
a solution such as buffer solution. As the buffer solution used for
preparing such suspension, any one usually used in this field is adopted
without specific limitation, and usually one having buffering action at
pH 5.0 to 10.0, preferably around neutral pH of pH 6.5 to 8.5, for
example, phosphate buffer, Tris buffer or Good buffer is preferable. In
this connection, depending on the characteristics of the insoluble micro
particles, some one has a tendency to make aggregation naturally by
leaving in suspended condition. In such case, the use of a mildly
alkaline buffer solution such as glycine buffer or boric acid buffer for
the preparation of suspension is far more preferable from a standpoint of
storage stability. In addition, the concentration of buffering agent in
these buffers is selected appropriately from the range of usually 10 to
500 mM, preferably 10 to 300 mM. In this connection, in the
aforementioned reagent, for example, a stabilizing agent such as a sugar,
a protein and a surface activating agent, a salt such as NaCl and an
preservative substance and the like may be added within the range usually
used in this field.

[0027]When the anti-HABP antibody-supported carrier involved in the
present invention is suspended in an above described buffer solution, the
concentration of the anti-HABP antibody-supported carrier in the reaction
may be, while it may vary depending on the kind of anti-HABP antibody
used, set out to be the same concentration as described above, and may be
selected appropriately so as to be usually within a range from 0.1 to 500
μg/ml, preferably from 0.5 to 100 μg/ml.

[0028]Further, in the reagent comprising an anti-HABP antibody-supported
carrier involved in the present invention, an immunological reaction
accelerator (agglutination reaction accelerator) (for example,
polyethylene glycol and polyvinyl alcohol) may coexist at the
concentration range usually used in this field, and even under
coexistence of such agglutination reaction accelerator, the appearance of
nonspecific turbidity of denatured protein constituent in the measuring
reagent, which is caused by some sort of factor, can be repressed or
reduced by the method of the present invention. In addition, a monomer or
a polymer used as an agglutination accelerator described in
JP-A-2002-365296 may be contained as an agglutination accelerator in the
above-described reagent, and the concentration range thereof may be
selected according to the value described in JP-A-2002-365296. In this
connection, the aforementioned monomer or polymer may be prepared
according to the method described in the above patent application.

[0029]The reagent kit for the measurement of hyaluronic acid involved in
the present invention is to be used for performing such measurement
method as described above of the present invention, and preferable
embodiments of constituent elements and specific examples are as
described above.

[0030]As to a sample involved in the present invention, any sample
containing hyaluronic acid may be adopted, and specifically, it includes,
for example, body fluid such as blood, plasma, serum, synovial fluid,
pleural fluid, lymph fluid, spinal fluid and urine, and as preferable
sample among them, serum, urine and the like are included.

[0031]Some examples exemplifying the present invention are described more
specifically below; however, the scope of the present invention should
not be limited thereto.

[0034]In the next step, the suspension of anti-HABP monoclonal
antibody-supported latex particles was centrifuged at 15000 rpm for 15
minutes. After the supernatant solution was removed, 1 ml of 50 mM boric
acid buffer (containing 2.5% BSA, pH 7.3) was added to the pellet at the
bottom of the tube for centrifugation. After that, the pellet was
resuspended by ultrasonication for 1 minute under cooling with ice, then,
was centrifuged at 15000 rpm for 15 minutes. After the supernatant
solution was removed, 1 ml of 50 mM boric acid buffer (containing 2.5%
BSA, pH 7.3) was added to the pellet at the bottom of the tube for
centrifugation. After that, the pellet was resuspended by ultrasonication
for 1 minute under cooling with ice. Further, the suspension was
incubated with stirring at room temperature for 120 minutes and the area
of the latex particle surface, on where antibody has not been supported,
was coated with BSA.

[0035]After that, the suspension was centrifuged at 15000 rpm for 15
minutes. After the supernatant solution was removed, 1 ml of 50 mM boric
acid buffer (containing 0.5% BSA, pH 7.3) was added to the pellet at the
bottom of the tube for centrifugation. Then, the pellet was resuspended
by ultrasonication for 1 minute under cooling with ice, and diluted 3.33
times with 50 mM boric acid buffer (containing 0.5% BSA, pH 7.3). This
solution was defined as second test solution.

(2) Measurement of Standard Hyaluronic Acid

1. Preparation of a Standard Solution of Hyaluronic Acid

[0036]Potassium hyaluronate (product of Wako Pure Chemical Industries,
Ltd.) was diluted with 50 mM phosphate buffer (pH 7.0) so as to make
solutions in concentration of 10, 100 and 1000 ng/ml, and used as
standard solutions of hyaluronic acid.

2. Measurement of Hyaluronic Acid

[0037]The quantity of hyaluronic acid in the standard solutions of
hyaluronic acid prepared in the above section 1 was measured under the
condition as shown below using a fully automated measuring equipment
system (JEOL Ltd.: BM-8 Model).

[0038]Sample: 10 μl

[0039]First test solution: 90 μl

[0040]Second test solution: 30 μl

[0041]Measurement method: 2 point-end method

[0042]Dominant wavelength: 571 nm

[0043]The results obtained were shown in Table 1. In this connection, the
value indicated in the table was the value increased 10000 times of that
obtained by subtraction of blank value (value obtained when hyaluronic
acid concentration is zero) from measured value.

[0044]To a 2 ml volume polycarbonate tube for centrifugation, 900 μl of
50 mM TAPS buffer solution (pH 8.0), 100 μl of carbonic acid latex
particle solution (10 wt %; carbonic acid latex particles: 200 nm in
diameter; carbonic acid content: 0.3 meq/g) and 50 μl of 10 mg/ml
aqueous solution of water soluble carbodiimide (WSC, product of Dojindo
Laboratories) were added, and then left standing for 10 minutes to
activate carboxyl group on the surface of latex particles. After that,
276 μl of 1.45 mg/ml HABP solution in ASES buffer solution (50 mM, pH
6.5) was added, and incubated with stirring at room temperature for 120
minutes. Additionally, 250 μl of boric acid buffer (50 mM, pH 7.3)
containing 2.5% BSA was added to the reaction solution and incubated with
stirring at room temperature for 60 minutes, and followed by incubation
at 5° C. for overnight. After that, the reaction solution was
centrifuged at 18000 rpm for 20 minutes. After the supernatant solution
was removed, 1 ml of 50 mM boric acid buffer (containing 0.5% BSA, pH
7.3) was added to the pellet at the bottom of the tube for
centrifugation. Then, the pellet was resuspended by ultrasonication for 1
minute under cooling with ice, and the same procedure was repeated twice.
The solution obtained was defined as second test solution (1).

[0045]HABP-sensitized latex particle was prepared by the same procedure as
described in Comparative Example 1 (1), except that the carbonic acid
latex particle solution with 10 wt %, 210 nm in diameter and 0.5 meq/g of
carbonic acid content was used. The solution obtained was defined as
second test solution (2).

In the next step, the reaction solution was centrifuged at 15000 rpm for
15 minutes. After the supernatant solution was removed, 1 ml of 50 mM
boric acid buffer (containing 2.5% BSA, pH 7.3) was added to the pellet
at the bottom of the tube for centrifugation. After that, the pellet was
resuspended by ultrasonication for 1 minute under cooling with ice. Then,
the suspension was centrifuged at 15000 rpm for 15 minutes. After the
supernatant solution was removed, 1 ml of 50 mM boric acid buffer
(containing 2.5% BSA, pH 7.3) was added to the pellet at the bottom of
the tube for centrifugation. Further, the pellet was resuspended by
ultrasonication for 1 minute under cooling with ice. Then the suspension
was incubated with stirring at room temperature for 60 minutes, and
followed by incubation at 5° C. for overnight.After that, the
suspension was centrifuged at 15000 rpm for 15 minutes. After the
supernatant solution was removed, 1 ml of 50 mM boric acid buffer
(containing 0.5% BSA, pH 7.3) was added to the pellet at the bottom of
the tube for centrifugation. Then, the pellet was resuspended by
ultrasonication for 1 minute under cooling with ice. The solution
obtained was defined as second test solution (3).

[0047]To a 2 ml volume polycarbonate tube for centrifugation, ml of
anti-HABP monoclonal antibody-sensitized latex particle solution prepared
in Example 1 and 33.5 μl of 896 μg/ml HABP aqueous solution were
added, and incubated with stirring at room temperature for 120 minutes,
and additionally incubated at 5° C. for 2 days. Then, the
resultant reaction solution was centrifuged at 15000 rpm for 15 minutes.
After the supernatant solution was removed, 1 ml of 50 mM boric acid
buffer (containing 0.5% BSA, pH 7.3) was added to the pellet at the
bottom of the tube for centrifugation. Then, the pellet was resuspended
by ultrasonication for 1 minute under cooling with ice. The solution
obtained was defined as second test solution (4).

(5) Comparison of Calibration Curve of Reagents for the Measurement of
Hyaluronic Acid Using Each Latex Particle Solution

[0048]The quantity of hyaluronic acid in the standard hyaluronic acid
solutions was measured by the same method as (2) of Example 1, except
that 100 mM HEPES buffer solution (containing 0.1% BSA and 1% NaCl,
pH7.0) was used as first test solution and the second test solutions (1)
to (4) were diluted by 3.33 times with 50 mM boric acid buffer
(containing 0.5% BSA, pH 7.3) as second test solution.

[0049]The obtained results were shown in Table 1 together with the result
of Example 1. In this connection, the value indicated in the table was
the value increased 10000 times of that obtained by subtraction of blank
value (value obtained when hyaluronic acid concentration is zero) from
measured value.

[0050]As is clear from the results shown in Table 1, when the
HABP-sensitized latex particle prepared either by chemical bond (second
test solution (1) and (2) in Table 1) and by physical adsorption (second
test solution (3) in Table 1) were used, agglutination of latex particles
corresponding to the concentration of hyaluronic acid was not observed,
and thus accurate measurement could not be performed. In addition, no
significant difference in the measurement was observed, between the case
when a test solution containing the particle, wherein HABP is
preliminarily bound to anti-HABP monoclonal antibody-supported latex
particle, was used as second test solution (second test solution (4) in
Table 1) and the case when a solution containing HABP was used as first
test solution and a solution containing anti-HABP monoclonal
antibody-supported latex particle was used as second test solution
(second test solution of Example in Table 1), and thus, it was confirmed
that, by the use of these methods, a highly accurate measurement of
hyaluronic acid can be performed.

Experimental Example 1

Comparison of Temporal Stability of Test Solutions

[0051]The first test solution and the second test solution were stored at
30° C. for 1 month, and then the quantity of hyaluronic acid was
measured by the same procedure as described in Example 1 (2) using these
stored test solutions. In addition, both the first test solution and the
second test solution (4) used in Comparative Example 1 were stored at
30° C. for 1 month, and then the quantity of hyaluronic acid was
measured by the same procedure as described in Comparative Example 1 (5)
using these stored test solutions.

[0052]The results obtained were shown in Table 2. In this connection, the
absorbance value indicated in the table was the value increased 10000
times of that obtained by subtraction of blank value (value obtained when
hyaluronic acid concentration is zero) from measured value. In addition,
absorbance retention was expressed as the percentage of the value
obtained by dividing the absorbance measured after incubation for 1 month
by the absorbance measured before incubation.

[0053]As is clear from the results shown in Table 2, when the second test
solution (4), namely, a test solution containing the particle, wherein
HABP is preliminarily bound to anti-HABP monoclonal antibody-supported
latex particle, was used as second test solution, the absorbance after
passing for 1 month, as compared to that of before passage, has decreased
obviously. On the other hand, when test solutions in Example 1 were used,
namely, a solution containing HASP was used as first test solution and a
solution containing anti-HABP monoclonal antibody-supported latex
particle was used as second test solution, the absorbance retention was
80% or more, and the stability of the reagent was found significantly
high compared to that observed for the second test solution (4). Thus, it
was confirmed that, by the use of a reagent kit of the present invention,
a highly accurate measurement of hyaluronic acid can be performed even if
the reagents are stored for long time.